Developmental and age-dependent plasticity of GABA_A receptors in the mouse colon: Implications in colonic motility and inflammation

Abstract

Lifelong functional plasticity of the gastrointestinal (GI) tract is essential for health, yet the underlying molecular mechanisms are poorly understood. The enteric nervous system (ENS) regulates all aspects of the gut function, via a range of neurotransmitter pathways, one of which is the GABA-GABA_A receptor (GABA_AR) system. We have previously shown that GABA_A receptor subunits are differentially expressed within the ENS and are involved in regulating various GI functions. We have also shown that these receptors are involved in mediating stress-induced colonic inflammation. However, the expression and function of intestinal GABA_ARs, at different ages, is largely unexplored and was the focus of this study. Here we show that the impact of GABA_AR activation on colonic contractility changes from early postnatal period through to late adulthood, in an age-dependant manner. We also show that the highest levels of expression for all GABA_AR subunits is evident at postnatal day (P) 10 apart from the α3 subunit which increased with age. This increase in the α3 subunit expression in late adulthood (18 months old) is accompanied by an increase in the expression of inflammatory markers within the mouse colon. Finally, we demonstrate that the deletion of the α3 subunit prevents the increase in the expression of colonic inflammatory markers associated with healthy ageing. Collectively, the data provide the first demonstration of the molecular and functional plasticity of the GI GABA_AR system over the course of a lifetime, and its possible role in mediating the age-induced colonic inflammation associated with healthy ageing.

Introduction

The integrity of the mammalian gastrointestinal tract (GIT) is fundamental for nutrition and barrier function over the course of a lifetime. The GIT exhibits remarkable plasticity during development and the ageing process, despite the changing nutritional needs of the individual, or the local environment in the form of microbiota and immune function (Salles, 2007). Nevertheless, ageing is associated with a general decline in intestinal function, manifesting in motility disorders such as constipation, or altered immune function, such as inflammation (Rao and Go, 2010). However, the molecular mechanisms underlying this age-related GI plasticity and the ensuing associated disorders have yet to be fully understood (Saffrey, 2014). Age-specific changes in the intrinsic nervous system of the GIT, namely the enteric nervous system (ENS) and its associated neurotransmitters systems are most likely central to lifelong GI health (Saffrey, 2013).

The ENS is integral to all aspects of coordinated GI function (Furness, 2008; Di Nardo et al., 2008). It is a large collection of neurochemically diverse neurons (Furness, 2000; Noorian et al., 2011) located within the muscle wall of the GIT. These neurons form intricate cellular networks with neuronal and non-neuronal cell-types and provide the intrinsic neural control of virtually all GI functions such as peristalsis (Grider, 1989), secretion (Riegler et al., 2000), barrier function (Neunlist et al., 2003; Toumi et al., 2003) and local immune function (Goyal and Hirano, 1996; Furness, 2006; Schneider et al., 2001). Despite our considerable insight into ENS-mediated GI function in adulthood, relatively less is known about how this differs throughout the process of ageing, from early postnatal days to late adulthood. The most consistent finding in the aged ENS is altered local immune function (Man et al., 2014) and degeneration of neurochemically-distinct cell-types (Saffrey, 2013; Camilleri et al., 2008; Bernard et al., 2009). However, there is considerably less known about the age-related changes in the neurotransmitter receptor systems through which different ENS neurons mediate their effects on the GIT. One of these neurotransmitter-receptor systems, the GABA-GABA_AR system has been shown to be involved in virtually all ENS-mediated GI functions (Krantis, 2000).

GABA_ARs are chloride permeable integral membrane ion channels composed of five interacting subunit proteins which mediate the rapid effects of the neurotransmitter GABA (Farrant and Nusser, 2005). While only five subunits are required to form a functional receptor, up to nineteen molecularly distinct GABA_AR subunits (α1–6; β1–3; γ1–3; δ; ε; ρ) have been identified, which underpin the expression of ~20–30 main distinct GABA_AR isoforms (Olsen and Sieghart, 2009). Within the central nervous system, these receptor subtypes display a differential regional expression or cellular location (Hortnagl et al., 2013) and exhibit specific physiological (Farrant and Nusser, 2005) and pharmacological properties (Rudolph and Knoflach, 2011). Previous studies in adult animals indicate that the GABA-GABA_AR system directly alters the excitability of ENS neurons (Cherubini and North, 1984), spontaneous colonic contractility (Bayer et al., 2002; Bayer et al., 2003; Seifi et al., 2014) and GI motility (Tonini et al., 1989a; Tonini et al., 1989b). However, the expression and functional plasticity of this neurotransmitter system at different maturational stages of the colon, is largely unexplored. Furthermore, we have recently reported that different GABA_AR subtypes have contrasting effects on stress-induced colonic inflammation (Seifi et al., 2018a). Given the consistent finding of altered immune function in the elderly, that may give rise to colonic inflammation (Ogra, 2010), the question arises whether GABA_ARs may be associated. In the current study, we show that the force and frequency of native spontaneous colonic contractions change significantly during early postnatal development stages. Furthermore, colonic GABA_AR expression changes dramatically in a subunit and age-dependent manner. Finally, the deletion of the GABA_AR α3 prevents age-related colonic inflammation.